Method for determination of the amount of either phosphotyrosine or phosphoserine in a protein

A method for determining the amount of phosphotyrosine in a protein comprising:

• a) reacting a protein with base at pH 14, to remove the phosphates from phosphoserine and phosphothreonine residues;
• b) acidifying said reaction mixture to pH about 3.5;
• c) reacting said acidified peptide with an alkylating agent;
• d) acidifying said reaction mixture to pH about 1.5 to form an acidified phosphonodiester of a phosphotyrosine;
• e) reacting said acidified phosphonodiester of a phosphotyrosine with a fluorescent tag;
• and
• f) quantitating the amount of said fluorescent tag.

A method for determining the amount of phosphotyrosine in a protein comprising:

• a) reacting a protein with an electrophilic reagent under appropriate conditions to react SH, NA.sub.2, OH and COOH groups;
• b) reacting a phosphotyrosine moiety in said protein with a species capable of electrophilic aromatic substitution;
• c) reacting said substituted aromatic with a fluorescent nucleophile;
• and
• d) determining the amount of said fluorescent nucleophile.

A method for determining the amount of phosphotyrosine in a protein comprising:

• a) reacting a protein with a base to remove the phosphates from phosphoserine and phosphothreonine residues;
• b) reacting said protein of step a) with a fluorophor;
• and
• c) reacting said fluorophorylated protein with a free radical;
• and
• d) quantitating the release of fluorophosphate.

A method for mapping the phosphorylation state of serine and tyrosine residues in a protein comprising:

• a) determining the amino acid sequence of a protein;
• b) fragmenting said protein;
• c) determining the presence of phosphoserine and phosphotyrosine residues in said fragments;
• and
• d) correlating the presence of phosphoserine and phosphotyrosine residues with the sequence of said protein.